Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer related death worldwide. The survival rate of HNSCC patients has not been significantly improved over the past 30 years with conventional therapies. More than 80% of HNSCC express high levels of epidermal growth factor receptor (EGFR), which correlate with poor prognosis. Targeting EFGR signaling with tyrosine kinase inhibitors (TKIs) or antibodies has emerged as a promising approach for treating cancers including HNSCC. The antitumor effects of EGFR blockade are thought to involve multiple mechanisms, including induction of apoptosis, whose underlying mechanism is not well understood. We and others previously identified the BH3- only Bcl-2 family member PUMA (p53 unregulated modulator of apoptosis) as an essential mediator of DNA damage-induced apoptosis. Our recent preliminary data showed that PUMA fails to be induced by conventional chemotherapeutic agents, but is induced by clinically approved EGFR targeting agents, gefitinib, erlotinib and cetuximab, in most HNSCC cell lines. PUMA induction is associated with EGFR TKI sensitivity. Knockdown of PUMA by small interference RNA (siRNA) and small hairpin RNA (hsRNA) suppressed gefitinib- induced apoptosis in HNSCC cells. Furthermore, EGFR TKIs induce PUMA through the effects on the p53 family members p63 and p73, but not p53 itself. These observations led us to hypothesize that PUMA induction is an important mechanism of EGFR blockade-induced cytotoxicity in HNSCC cells. We propose to test this central hypothesis using cell culture, animal tumor models and clinical samples. In Aim 1, we will delineate the molecular mechanisms of PUMA induction by EGFR inhibition in HNSCC cells. We will focus on the role of p73 and p63 in the transcriptional regulation of PUMA. In Aim 2, we will investigate the role of PUMA in EGFR inhibition-induced apoptosis in HNSCC cells. We will focus on the role and mechanism of PUMA in the regulation of mitochondrial integrity and other Bcl-2 family proteins. In Aim 3, we will determine whether PUMA levels modulate the therapeutic responses to EGFR targeted therapy in cell culture, xenograft models and clinical samples. PUBLIC HEALTH RELEVANCE: Head and neck squamous cell carcinoma (HNSCC) is a significant cause of cancer related death in the United States. This project seeks to understand the mechanisms by which a novel class of anticancer agent, epidermal growth factor receptor (EGFR) antagonists, exerts their antitumor effects in HNSCC. The results could be useful for developing improved strategies and agents for treatment of HNSCC and possibly other cancers with EGFR overexpression.